Volume 24, Issue 3, Pages 687-700 (November 1999) Essential Roles in Synaptic Plasticity for Synaptogyrin I and Synaptophysin I  Roger Janz, Thomas C Südhof, Robert E Hammer, Vivek Unni, Steven A Siegelbaum, Vadim Y Bolshakov  Neuron  Volume 24, Issue 3, Pages 687-700 (November 1999) DOI: 10.1016/S0896-6273(00)81122-8

Figure 1 Generation of Synaptogyrin I Knockout Mice (A) Gene targeting strategy. A genomic clone containing two exons of the synaptogyrin gene (putative exons II and III encoding residues 34–112 and 113–161, respectively; top line drawing) was used to construct a targeting vector that deletes exon II (middle). The targeting vector contains a central neomycin resistance gene cassette (neo) for positive selection. The neo cassette is flanked by long and short arms and two copies of the HSV thymidine kinase gene (2xTK) for negative selection. Homologous recombination shortens a 12 kb genomic BamHI wild-type fragment to a 9 kb knockout fragment, as analyzed with a probe from outside the targeting vector (probe). The locations of BamHI (B) and EcoRI sites (E) are indicated. (B) Southern blot analysis of BamHI-digested genomic DNA from offspring of crosses between heterozygous synaptogyrin mutant mice. The wild-type allele generates an ≈12 kB BamHI fragment and the mutant allele an ≈9 kB fragment. Genotypes are identified on top of the blot (+/+, wild-type; +/−, heterozygous; −/−, homozygous mutant mice). (C) Immunoblot analysis of total brain protein of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) synaptogyrin mutant mice using antibodies to synaptogyrin, synaptophysin, and synaptobrevin II. Positions of molecular weight markers are indicated on the left. Note the decrease in synaptogyrin levels in the heterozygotes. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 2 Breeding Strategy of Synaptogyrin I and Synaptophysin I Knockout Mice In the F0 generation, five homozygous mutant synaptogyrin I mice were mated with five homozygous mutant synaptophysin I mice to yield a generation of heterozygous mice (F1; 13 females, 10 males). These mice were interbred as indicated under avoidance of brother/sister matings to obtain an F2 generation that included double knockout mice, single knockout mice, and wild-type mice in the indicated numbers in addition to single and double heterozygotes. The heterozygotes were discarded. The homozygotes were used for further interbreeding with at least 10 independent founder mice per genotype. Brother/sister matings were again excluded as indicated for double knockout mice (black full symbols). Most analyses were performed with mice from the F4 generation. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 3 Immunohistochemical Analysis of the Hippocampus from Wild-Type and Double Knockout Mice Lacking Synaptogyrin I and Synaptophysin I Pictures show sagittal sections of wild-type (A, C, and E) and mutant brains (B, D, and F). Sections were stained with antibodies to synaptogyrin (A and B), synaptoporin (C and D), and rab 3A (E and F). Immunoreactivity was visualized using the peroxidase-anti-peroxidase method with heavy metal enhancement. The dentate gyrus (DG), CA1 region (CA1), CA3 region (CA3), and cortex (C) are identified in (A). The calibration bar in (A) corresponds to 200 μm and applies to all panels. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 4 Glutamate Release from Synaptosomes (A) Synaptosomes from wild-type (WT) and synaptogyrin/synaptophysin double knockout mice (DKO) were loaded with 3H-glutamate and superfused with Krebs-bicarbonate buffer under continuous monitoring of glutamate efflux. Release was induced by 30 s pulses of 25 mM KCl (K+) after 3 min superfusion and of 0.5 M sucrose after 10 min superfusion. Fractions were collected in 1 min intervals, and the fractional release rate of 3H-glutamate was calculated as percent of the total glutamate present at that time point in the synaptosomes. The graph shows a representative experiment using two mice of each genotype in duplicate measurements. Error bars, SEM. (B) Summary graph of 3H-glutamate release in wild-type and double knockout mice from four experiments. Release observed in the second minute of collection was taken as basal release. KCl (K+) and sucrose-induced peak release was calculated as the sum of the two peak release rates induced by the stimulus. The data are mean ± SEM. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 5 Paired-Pulse Facilitation in Synaptophysin I, Synaptogyrin I, and Double Knockout Mice Pairs of presynaptic stimuli were delivered 8–12 times for each interstimulus interval. The resulting pairs of field EPSPs were averaged for each slice. The EPSP for the second pulse was normalized to the EPSP of the first pulse. Normalized EPSPs are plotted as a function of the interstimulus interval (in ms) for synaptogyrin-deficient (A), synaptophysin-deficient (B), and double knockout mice (C). Each experiment was carried out by comparing wild-type and the respective knockout strains (wild-type mice, open circles; knockout mice, solid circles). The numbers of mice and slices analyzed are indicated on each graph. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 6 Posttetanic Potentiation in Synaptophysin I, Synaptogyrin I, and Double Knockout Mice PTP was induced by a single 1 s stimulus train of 100 Hz, with field EPSPs recorded every 3 s. Normalized EPSPs obtained from synaptophysin knockout (A), synaptogyrin knockout (B), and double knockout mice (C) are plotted as a function of time. Each experiment was performed with an internal wild-type control as shown (wild-type mice, open circles; knockout mice, solid circles), with the number of slices and animals used indicated. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 7 Long-Term Potentiation in Synaptophysin I, Synaptogyrin I, and Double Knockout Mice (A) through (C) exhibit results of LTP experiments in synaptophysin-deficient (A), synaptogyrin-deficient (B), and double knockout mice (C) in which LTP was induced by two stimulus trains (100 Hz for 1 s) spaced 20 s apart. Extracellular field recordings were used to sample EPSPs every 30 s. In the experiments shown in (D), LTP was induced by a voltage pairing protocol using whole-cell recording. Each experiment was performed with the respective knockouts and wild-type mice (wild-type mice, open circles; knockout mice, solid circles). Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 8 Synaptic Depression during High-Frequency Repetitive Stimulation in Synaptophysin I, Synaptogyrin I, and Double Knockout Mice EPSPs were recorded from slices in a bath solution with 50 μM D-APV under baseline conditions (30 responses sampled every 6 s) in normal external Ca2+ (2.5 mM, [A]–[C] and [E]) and elevated external Ca2+ (5.0 mM, [D and F]). After baseline recordings, 300 pulses were applied at 70 ms intervals (14 Hz). The second arrow shows when the test rate of stimulation (once every 6 s) was resumed. Normalized successive synaptic responses are plotted as a function of stimulus number for wild-type and knockout mice with the indicated number of slices and animals (wild-type mice, open circles; knockout mice, solid circles). (E) and (F) depict the recovery phase from synaptic depression from (C) and (D) at an expanded time scale. Following the high-frequency train used to induce depression, the amplitude of the field EPSP was measured after return to the initial low rate of stimulation (once per 30 s). The amplitude of the EPSPs was normalized by the size of the EPSPs before the high-frequency train. The EPSP amplitude recovers after 30–90 s. The mutant mice show a significant slowing of the time course of recovery from depression in both normal (2.5 mM) and elevated extracellular Ca2+ (5.0 mM). Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 9 Rate of MK-801 Blockade Is Not Altered upon Deletion of Both Synaptophysin I and Synaptogyrin I but Is Enhanced by Increased Release Probability (A) Rate of MK-801 block is not altered in double knockout mice. The progressive block by MK-801 of the NMDA receptor EPSC is shown under whole-cell recording conditions from CA1 pyramidal neurons during stimulation of the Schaffer collateral input. Averaged data from wild-type mice (open circles; data from eight slices from four mice) and double knockout mice (solid circles; data from seven slices from four mice) (P = 0.48 for pulses 1–30, F [1,13] = 0.525, two-way ANOVA). (B) Rate of MK-801 block is significantly enhanced by elevation of external Ca2+ from 2.5 mM (open circles) to 5.0 mM (filled circles) (P < 0.04 for pulses 1–30, F [1,13] = 5.9). (C) Rate of MK-801 block is significantly enhanced by application of adenosine antagonist. Open circles, absence of antagonist. Filled circles, presence of antagonist (three slices from three mice) (P < 0.005 for pulses 1–30, F [1,9] = 13.25). 40 μM MK-801 was applied to the slices in the absence of presynaptic stimulation. After a period ≥ 8 min, the Schaffer collateral input was stimulated once every 8 s. For each cell, EPSC amplitudes were normalized by the first EPSC. Data from all cells were then averaged. The same control data are shown in each panel (open symbols). Error bars show SEM. External solution contained 1.0 mM Mg2+ and 10 μM CNQX. Holding potential was −40 mV. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)

Figure 10 Tyrosine Phosphorylation of Synaptogyrin I and Synaptophysin I (A) Synaptogyrin I and synaptophysin I are in vivo substrates for c-src and fyn tyrosine kinases. COS cells were cotransfected with expression vectors encoding synaptogyrin (lanes 1, 4, and 7), synaptophysin (lanes 2, 5, and 8), or control plasmid (lanes 3 and 6) and c-src (lanes 3–5) or c-fyn protein kinase (6–8). COS cell proteins were analyzed by immunoblotting with phosphotyrosine antibodies. The synaptogyrin and synaptophysin bands on the blot were confirmed by immunoblotting (data not shown) and are identified by closed and open arrows, respectively. (B) Tyrosine-phosphorylated proteins in wild-type and double knockout mice. Crude synaptosomes (P2) and synaptic vesicles (LP2) prepared from wild-type (WT) and synaptophysin/synaptogyrin double knockout mice (DKO) were analyzed by immunoblotting using a phosphotyrosine-specific antibody. Bands corresponding to synaptophysin I and synaptogyrin I are marked with asterisks and arrows. Neuron 1999 24, 687-700DOI: (10.1016/S0896-6273(00)81122-8)